Three types of transmembrane proteins are expressed in the membrane of influenza type A virions and virus-infected cells. The hemagglutinin and neuraminidase are glycoproteins with large ectodomains of ˜510 and ˜420 amino acids, respectively. Hemagglutinin is assembled as homotrimers and neuraminidase as homotetramers forming a dense layer of 13-14 nm long, rod-shaped surface projections on the viral membrane and at cellular sites of virus maturation. Current influenza virus vaccines aim at inducing a strong antibody response to these glycoproteins, particularly the hemagglutinin, as such antibodies are well-known to be highly protective against infection. The problem is that influenza type A virus has a high propensity for changing the determinants recognized by these protective antibodies, which necessitates repetitive vaccination with updated vaccine strains that reflect these antigenic changes. By contrast, the third viral transmembrane protein, matrix protein 2 (M2), contains an ectodomain (M2e) that is conserved amongst human influenza virus strains. Broad protective immunity against influenza type A virus infection using M2 has been investigated (Slepushkin, et al. (1995) Vaccine 13:1399-1402; Frace, et al. (1999) Vaccine 17:2237-44; Neirynck, et al. (1999) Nature Med. 5:1157-63; Okuda, et al. (2001) Vaccine 19:3681-91).
M2 is a 97 amino acid long transmembrane protein of influenza type A virus (Lamb, et al. (1981) Proc. Natl. Acad. Sci. USA 78:4170-4174; Lamb, et al. (1985) Cell 40:627-633). The mature protein forms homotetramers (Holsinger and Lamb (1991) Virology 183:32-43; Sugrue and Hay (1991) Virology 180:617-624) that have pH-inducible ion channel activity (Pinto, et al. (1992) Cell 69:517-528; Sugrue and Hay (1991) supra). M2-tetramers are expressed at high density in the plasma membrane of infected cells but are relatively excluded from sites of virus maturation and therefore are incorporated only at a low frequency into the membrane of mature virus particles (Takeda, et al. (2003) Proc. Natl. Acad. Sci. USA 100:14610-14617; Zebedee and Lamb (1988) J. Virol. 62:2762-2772). The sequence of the 24 amino acid long ectodomain of M2 (M2e) has remained conserved amongst human epidemic virus strains (Macken, et al. (2001) In Options for the Control of Influenza. IV. Osterhaus, et al. (ed.), p. 103-106. Elsevier Science, Amsterdam). The majority of human epidemic strains isolated since 1918 share the same M2e protein sequence. Further, several studies in mice have shown that M2e-specific antibodies restrict influenza virus replication and reduce morbidity and mortality (Fan, et al. (2004) Vaccine 22:2993-3003; Liu, et al. (2004) Immunol. Lett. 93:131-136; Mozdzanowska, et al. (2003) J. Virol. 77:8322-8328; Neirynck, et al. (1999) supra; Treanor, et al. (1990) J. Virol. 64:1375-1377). Moreover, in ferrets, the animal model considered most prognostic for human influenza, protective activity of M2e-specific immunity has been demonstrated (Fan, et al. (2004) supra) and sera from rhesus monkeys immunized with a M2e-carrier conjugate have been shown to exhibit protective activity upon transfer into mice (Fan, et al. (2004) supra). Thus, with the exception of a study in pigs, which indicated that M2e-specific immunity may enhance rather than ameliorate disease (Heinen, et al. (2002) J. Gen. Virol. 83:1851-1859), evidence from animal models shows that M2e-specific immunity is capable of providing a significant level of protection that is directed against a remarkably conserved viral target.
The low degree of structural variation in M2e is certainly in part attributable to constraints resulting from its genetic relation to M1, the most conserved protein of the virus (Ito, et al. (1991) J. Virol. 65:5491-5498). M2 is encoded by a spliced RNA of the viral gene segment 7, which codes also for M1 (Lamb, et al. (1985) supra). The splicing event removes most of the nucleotides that code for M1 (nt 27-714) except the 26 most 5′ and 42 most 3′ nucleotides (Lamb, et al. (1985) supra). Thus, nucleotides 1-68 of M2 which encode essentially the entire M2e are bicistronic, from 1-26 in the same and from 27-68 in a different reading frame. This genetic relation between M2e and M1 can be expected to substantially restrict the degree of variability in M2e. An additional factor that may contribute to the low degree of change seen in M2e amongst human influenza virus strains could be the absence of M2e-specific antibodies and thus pressure for change. A small study of 17 paired human sera obtained during the acute and convalescent phase of natural infection found that M2-specific antibodies were absent from acute sera and became detectable in only six of the convalescents (Black, et al. (1993) J. Gen. Virol. 74 (Pt 1):143-146). This was in contrast to nucleoprotein-specific antibody titers, which increased in 15 of 17 convalescent sera, thus confirming recent influenza infection of the donors (Black, et al. (1993) supra). Another study found no difference in M2e-specific antibody titers in two larger groups of unpaired sera, one positive and the other negative for virus-specific antibodies (Liu, et al. (2003) FEMS Immunol. Med. Microbiol. 35:141-146). These data suggest that while infection in humans can result in a measurable antibody response to M2, the response is not generated consistently and is small and of short duration. Similar observations have been made in the mouse model where two repetitive infections with virus strains that shared the same M2e induced only low titers of M2e-specific antibodies (Mozdzanowska, et al. (2003) Vaccine 21:2616-2626). Since M2 is a minor component (<0.5%) of purified virus (Zebedee and Lamb (1988) J. Virol. 62:2762-2772), inactivated influenza virus vaccines presently being used would not be expected to induce M2e-specific immunity either.
M2e-specific monoclonal antibody 14C2 does not prevent virus infection in vitro but reduces virus yield and plaque size when incorporated into the culture medium or agar overlay (Zebedee and Lamb (1988) supra; Hughey, et al. (1995) Virology 212:411-21). Not all M2e-specific antibodies display this activity (Hughey, et al. (1995) supra) and not all virus strains are susceptible to it (Zebedee and Lamb (1988) supra). In vivo, passive monoclonal antibody 14C2 similarly decreases virus growth (Treanor, et al. (1990) J. Virol. 64:1375-7) and is effective also against PR8 (Mozdzanowska, et al. (1999) Virology 254:138-46), which is not susceptible to antibody-mediated growth restriction in vitro (Zebedee and Lamb (1988) supra; Mozdzanowska, et al. (1999) supra), indicating that antibody-mediated virus growth-inhibition occurs through distinct mechanisms in vitro and in vivo.
It has now been found that a multiple antigenic agent containing M2e linked to helper T cell determinants is an effective vaccine for inducing virus protection. M2e-MAAs together with cholera toxin (CT) and a synthetic oligodeoxynucleotide (ODN) with a stimulatory CpG motif induces strong M2e-specific antibody titers in serum of mice and results in significant protection against influenza virus challenge.